Analysis tools for single-monomer measurements of self-assembly processes

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin

Influence of protein kinases on the osmosensitive release of taurine from cerebellar granule neurons

The role of phosphorylation events on the activation and modulation of the osmosensitive (3)H-taurine release (OTR) was examined in cultured cerebellar granule neurons (CGN) stimulated with 30% hyposmotic solutions. OTR was not decreased when [Ca(2+)](i) rise evoked by hyposmolarity was prevented by EGTA-AM (50 microM) or depleted by treatment with 1 microM ionomycin in Ca(2+)-free medium. Accordingly, OTR was not inhibited by Ca(2+)-dependent signaling events. The calmodulin (CAM) blocker W-7 (50 microM) potentiated OTR while the Ca(2+)/CAM kinase blocker KN-93 (10 microM) was without effect. Blockade of PKC by H-7, H-8 (50 microM) and Gö6976 (1 microM), as well as activation by phorbol myristate acetate (PMA) (100 nM) did not influence OTR, but chronic treatment to down regulate PKC decreased it by 30%. Forskolin (20 microM) and 8-BrcAMP (10 microM) did not change OTR. Protein tyrosine phosphorylation seems to be of crucial importance in the activation and modulation of OTR, as it was markedly inhibited (90%) by tyrphostine A23 (50 microM) and potentiated by the tyrosine phosphatase inhibitor ortho-vanadate (100 microM). The PI3 kinase blocker wortmannin 100 nM essentially abolished OTR but LY294002 (10-100 microM) was without effect. This difference may be accounted for PI3K isoforms in neurons with different sensitivity to the blockers. Alternatively, the effect of wortmannin may be exerted not in PI3 kinase but instead on phospholipases, which are also sensitive to this blocker. The hyposmotic stimulus induced activation of Erk1/Erk2, but blockade of this effect by PD 98059 (50 microM) only marginally decreased OTR suggesting that the Erk1/Erk2 is an epiphenomenon, not directly involved in OTR activation

Effects of Hofmeister Ions on the alpha-Helical Structure of Proteins

The molecular conformation of proteins is sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. Here, we combine circular dichroism (CD), single-molecule Förster resonance energy transfer, and atomistic computer simulations to elucidate salt-specific effects on the structure of three peptides with large α-helical propensity. CD indicates a complex ion-specific destabilization of the α-helix that can be rationalized by using a single salt-free computer simulation in combination with the recently introduced scheme of ion-partitioning between nonpolar and polar peptide surfaces. Simulations including salt provide a molecular underpinning of this partitioning concept. Furthermore, our single-molecule Förster resonance energy transfer measurements reveal highly compressed peptide conformations in molar concentrations of NaClO(4) in contrast to strong swelling in the presence of GdmCl. The compacted states observed in the presence of NaClO(4) originate from a tight ion-backbone network that leads to a highly heterogeneous secondary structure distribution and an overall lower α-helical content that would be estimated from CD. Thus, NaClO(4) denatures by inducing a molten globule-like structure that seems completely off-pathway between a fully folded helix and a coil state

Thalamo-hippocampal pathway regulates incidental memory capacity in mice

Incidental memory is affected by retention delay, and by memory load. Here the authors show that female and male mice process high memory load through different activation of thalamic-cortical pathways, that makes their incidental memory resistant to distraction and to memory decay, respectively

Thalamo-hippocampal pathway regulates incidental memory capacity in mice

Incidental memory can be challenged by increasing either the retention delay or the memory load. The dorsal hippocampus (dHP) appears to help with both consolidation from short-term (STM) to long-term memory (LTM), and higher memory loads, but the mechanism is not fully understood. Here we find that female mice, despite having the same STM capacity of 6 objects and higher resistance to distraction in our different object recognition task (DOT), when tested over 1 h or 24 h delays appear to transfer to LTM only 4 objects, whereas male mice have an STM capacity of 6 objects in this task. In male mice the dHP shows greater activation (as measured by c-Fos expression), whereas female mice show greater activation of the ventral midline thalamus (VMT). Optogenetic inhibition of the VMT-dHP pathway during off-line memory consolidation enables 6-object LTM retention in females, while chemogenetic VMT-activation impairs it in males. Thus, removing or enhancing sub-cortical inhibitory control over the hippocampus leads to differences in incidental memory

Chivosazole A Modulates Protein-Protein Interactions of Actin.

Actin is a protein of central importance for many cellular key processes. It is regulated by local interactions with a large number of actin binding proteins (ABPs). Various compounds are known to either increase or decrease the polymerization dynamics of actin. However, no actin binding compound has been developed for clinical applications yet because of selectivity issues. We provide a crystal structure of the natural product chivosazole A (ChivoA) bound to actin and show that-in addition to inhibiting nucleation, polymerization, and severing of F-actin filaments-it selectively modulates binding of ABPs to G-actin: Although unphysiological actin dimers are induced by ChivoA, interaction with gelsolin, profilin, cofilin, and thymosin-β4 is inhibited. Moreover, ChivoA causes transcriptional effects differing from latrunculin B, an actin binder with a different binding site. Our data show that ChivoA and related compounds could serve as scaffolds for the development of actin binding molecules selectively targeting specific actin functions